P858: FUNCTIONAL STUDIES IN MULTIPLE MYELOMA CELL LINESWITH OVEREXPRESSION OF NRAS Q61R MUTATION
Authors: Romano Liotti, Angelica Macauda, Rainer Will, Stefano Landi, Federica Gemignani, Federico Canzian
Journal: HemaSphere (2023)

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Multiple myeloma (MM) is one of the most common hematological malignancies, accounting for 20% of all newly diagnosed hematological cancers. The most recent data from Globocan show that in 2020, worldwide, the number of new cases was 159,985. Although the current 5-year survival rate is over 50% MM is still considered an incurable disease since most of the patients eventually relapse. Presence of RAS genes is reported in about 50% of newly diagnosed MM and about 75% of relapsed/refractory MM (rrMM). Interestingly, more than half of patients with RAS activation harbor activating mutations in NRAS or KRAS genes, most of which clustered into codons 12, 13, or 61 and associated with a shorter overall and progression-free survival. The NRAS Gln61Arg mutation (Q61R) with a frequency of 6.82% represents the most frequent point mutation in patients with MM. NRAS Q61R is a hotspot mutation that lies within a GTP-binding region of the NRAS protein resulting in an increased GTP-bound NRAS and leading to activation of MAPK signaling and cell transformation. The exact functional consequences of the NRAS Q61R mutation in the context of MM are not yet well known. Aims: Explore the transformations resulting from the overexpression of NRAS with the Q61R mutation in MM cell lines through functional studies. Methods: The human U266B1 MM cell line was stably transduced with an overexpression construct carrying the human NRAS Q61R hotspot mutation. The functional effects of the overexpression were assessed through cell migration and cell viability assays (respectively Transwell and celltitle glow) and compared with a control cell line with an empty vector and a control carrying no vector (U266B1 wild type). Results: Overexpression of the NRAS Q61R mutation induces remarkable changes in MM cell lines. Namely, it appears to have a negative effect, with reduced metabolic activity and reduced cell proliferation. Specifically, a 45% reduction in cell proliferation was observed in the cells overexpressing NRAS Q61R in comparison with the ones with the empty vector. The migration assays however revealed a more aggressive phenotypic behavior with increased tendency to migration and invasiveness. In particular, the overexpressing cells migrated 10 times more than the wild type cells and 3 times more than their control without vector (Figure 1). Summary/Conclusion: This study aimed to explore the functional effects of NRAS Q61R overexpression in MM cell lines. The results showed that overexpression of NRAS Q61R negatively affects cell proliferation but increases cell migration and invasiveness. These findings suggest mechanisms by which NRAS Q61R may play a significant role in the progression and aggressiveness of MM. Figure 1. Representative Transwell migration assay images (10× magnification)a-f a) U266B1 WT on fixed membrane with fluorescent dye DAPI b) visualization of U266B1 WT cell outlines c) U266B1 OE NRAS Q61R on fixed membrane with fluorescent dye DAPI d) visualization of U266B1 OE NRAS Q61R cell outlines e) U266B1 Ctrl Puromycin on fixed membrane with fluorescent dye DAPI f) visualization of U266B1 Ctrl Puromycin cell outlinesKeywords: Point mutation, Multiple myeloma